The acceptor specificities of bacterial nucleoside phosphotransferase were further investigated by phosphorylating various kinds of nucleoside analogues. The bacteria belonging to A group(5′-nucleotide former) specifically phosphorylated the primary alcohol at 5′-position of nucleosides and their analogues, such as adenine xyloside, psicofuranine and pseudouridine, while the others belonging to B group (3′(2′)-nucleotide former) the secondary alcohol at 3′(2′)-position. The phosphorylation at 5′-primary alcohol with the bacteria belonging to A group, however, was prohibited mainly by phosphoryl-or amino-radical at 3′-position, as observed in the case of 3′-nucleotide or amino-nucleoside (or puromycin), depending on the steric conformation around the 3′-position of acceptor. Besides, both types of nucleoside phosphotransferases were also able to phosphorylate nucleoside having a C-C-linkage between base and sugar moieties.